Sedimentation field flow fractionation is a versatile technique for the high resolution separation of a wide variety of particulates suspended in a fluid medium. The particulates including macromolecules in the 10.sup.5 to the 10.sup.13 molecular weight (0.001 to 1 .mu.m) range, colloids, particles, micelles, organelles and the like. The techniques is more explicitly described in U.S. Pat. No. 3,449,938, issued June 17, 1969 to John C. Giddings and U.S. Pat. No. 3,523,610, issued Aug. 11, 1970 to Edward M. Purcell and Howard C. Berg.
Field flow fractionation is the result of the differential migration rate of components in a carrier or mobile phase in a manner similar to that experienced in chromatography. However, in field flow fractionation there is no separate stationary phase as is in the case of chromatography. Sample retention is caused by the redistribution of sample components between the fast to the slow moving strata within the mobile phase. Thus, particulates elute more slowly than the solvent front.
Typically a field flow fractionation channel, consisting of two closely spaced parallel surfaces, is used wherein a mobile phase is caused to flow continuously through the gap between the surfaces. Because of the narrowness of this gap or channel (typically 0.025 centimeters (cm)) the mobile phase flow is laminar with a characteristic parabolic velocity profile. The flow velocity is the highest at the middle of the channel and essentially zero at the two channel surfaces. An external force field of some type (the force fields include gravitational, thermal, electrical, fluid cross flow and others described variously by Giddings and Berg and Purcell), is applied transversely (perpendicular) to the channel surfaces or walls. This force field pushes the sample components in the direction of the slower moving strata near the outer wall. The buildup of sample concentration near the wall, however, is resisted by the normal diffusion of the particulates in a direction opposite to the force field. This results in a dynamic layer of component particles, each component with an exponential-concentration profile. The extent of retention is determined by the particulates time average position within the concentration profile which is a function of the balance between the applied field strength and the opposing tendency of particles to diffuse.
In sedimentation field flow fractionation (SFFF), use is made of a centrifuge to establish the force field required for the separation. For this purpose a long, thin, annular belt-like channel is made to rotate within a centrifuge. The resultant centrifugal force causes components of higher density than the mobile phase to sediment toward the outer wall of the channel. For equal particle density, because of higher diffusion rate, smaller particulates will accumulate into a thicker layer against the outer wall than will larger particles. On the average, therefore, larger particulates are forced closer to the outer wall.
If now the fluid medium, which may be termed a mobile phase or solvent, is fed continuously from one end of the channel, it carries the sample components through the channel for later detection at the outlet of the channel. Because of the shape of the laminar velocity profile within the channel and the placement of particulates in that profile, solvent flow causes smaller particulates to elute first, followed by a continuous elution of sample components in the order of ascending particulate mass.
In order to reduce the separation times required using this technique, it is necessary to make the channels relatively thin as noted. This creates many problems because, in order to maintain a high degree of resolution of the separated components of the sample, the channel must maintain a constant thickness during operation even when subjected to large centrifugal forces. This is not easily accomplished, particularly if the weight of the channel elements are to be maintained at reasonably small values for use in the centrifuge. The inner radial wall of the channel tends to bow radially outward when subjected to centrifugal force.
If the inner channel wall thickness t is too great the wall tends to bow radially outward into channel when subjected to centrifugal force. This is due to the fact that the centrifugal force on the wall exceeds the counter fluid pressure force pushing radially inward on the wall. Likewise, if the wall thickness t is too thin the wall will bow radially inward, opening up the channel, since in this case the pressure loading exceeds the wall centrifugal or body force. The degree of bowing or wall deflecting increases as the square of the rotational speed.
This wall deflection produces a variable channel radial width W which in turn is produces a nonuniform flow profile across the axial or width dimension of the flow channel. This nonuniformity in flow tends to first spread a sample population due to the difference in velocity across the width of the channel and secondly creates a nonuniform retention across the axial height of the flow channel. Both problems tend to vary as functions of rotor speed. This nonuniformity tends to degrade results considerably.